This proposal is a competitive renewal (CA74305) that concerns the application of chemical approaches to the investigation of the mechanisms and effects of phosphorylation of cell signaling proteins. The importance of protein phosphorylation is becoming increasing well recognized in cell growth, differentiation and human diseases. However, a detailed knowledge is lacking of how protein kinases are regulated, how they recognize their substrates, and what the function of their phosphorylation events are. We plan to develop and apply three different chemical technologies to help to address these problems. First, we are using expressed protein ligation to investigate the biochemical, structural, and functional consequences of tail tyrosine phosphorylation of several important signaling enzymes (SHP-1, SHP-2, Src, Csk). In particular, how tall tyrosine phosphorylation of proteins affects the structure and function of these enzymes will be investigated. Second, we are developing a chemical rescue approach to complement mutant, catalytically defective protein tyrosine kinases with the long term goal of revealing novel aspects of their functions in cell signaling. The loss of a catalytically important arginine and its complementation by small diamino compounds will be studied using protein tyrosine kinase Csk as a paradigm system. Third, we are synthesizing, rationally designed, bisubstrate analog protein kinase inhibitors as structural and biological tools for several protein tyrosine and serine/threonine kinases. Structural features of these compounds that are critical for potency and specificity will be probed by synthetic modifications. The development and application of these chemical methods should ultimately provide a greater understanding of the roles of protein phosphorylation in cellular function in healthy and disease states. The new approaches to protein kinase inhibitors may ultimately provide lead agents for the treatment of cancer, immune system-related diseases, and cardiovascular pathologies.